1. Field of the Invention
The disclosure relates to a liquid chromatograph. Specifically, the disclosure relates to a two-dimensional liquid chromatographic analyzer having a plurality of separation columns and an analytical method.
2. Description of the Related Art
A liquid chromatograph is an apparatus configured to separate objective components in a sample by a separation column while feeding a mobile phase and to detect the components that flow out in an order of their separation by a detector such as a spectrophotometer so as to analyze the components of the sample.
For the liquid chromatograph, separation columns of various separation modes such as an ion-exchange mode, a normal phase mode, and a reversed phase mode are employed for separating ionic components, hydrophilic components, hydrophobic components, and the like in samples and the kind of the mobile phase varies depending on the separation mode.
For the mobile phase, for improving separation and shortening analysis time, there is generally used gradient elution in which two or more kinds of mobile phases are fed while changing a mixing ratio thereof.
On the other hand, there is also known a method of achieving an improvement in separation and shortening of analysis time by changing the temperature of the separation column. For example, in a reversed phase chromatograph in which, as a representative reversed phase separation column, an octadecylsilyl (ODS) group is used as a functional group, it is generally known that elution is fastened when the temperature of the separation column becomes high.
Japanese Patent No. 5362943 reports a simple and convenient drug metabolizing ability evaluation system in which a filling agent whose surface is covered with a polymer having hydration force that changes within the temperature range of 0 to 80° C. is used for a separation column and objective components are separated and measured by controlling the temperature of the separation column.
However, in the case where components in a biological sample such as serum are analyzed using a liquid chromatograph, a plurality of components are present as a mixture in the biological sample, and use of one column may result in insufficient separation or may take a long period of time for analysis.
In Japanese Patent No. 5362943, after the biological sample is pre-treated using a column for solid-phase extraction, objective components are analyzed through a separation column.
As a specific separation method, there is used a two-dimensional liquid chromatographic analytical method in which, using a flow channel switching valve, columns of two kinds of different separation modes are combined and the objective components separated by a first (first-dimensional) separation column are fractionated and then introduced into a second (second-dimensional) column to further separate them.
In the two-dimensional liquid chromatography, it is common to perform analysis using an ion exchange column as the first-dimensional column and a reversed phase column as the second-dimensional one.
In the first-dimensional ion exchange column, the objective components are separated by salt concentration or pH of a buffer solution as a mobile phase and the separated objective components are introduced into the second-dimensional reversed phase column. In the second-dimensional reversed phase column, further separation is performed using a mobile phase containing an organic solvent.
JP-A-2008-96455 describes examples of the two-dimensional liquid chromatographic analyzer in which the first-dimensional separation and the second-dimensional separation are performed.
The technique described in JP-A-2008-96455 is that, in order to perform replacement of the mobile phase in the system efficiently, the components separated in the first dimension are once trapped and concentrated by a trap column and the components trapped by the trap column are further separated and analyzed in the second dimension. As a configuration thereof, there has been reported a two-dimensional liquid chromatograph capable of analyzing even any components using fewest three trap columns.
JP-A-2008-96455 describes a two-dimensional liquid chromatograph comprising a first-dimensional separation flow channel for introducing a sample injected from a sample injection part into a separation column with a mobile phase for separation to separate the sample, three trap columns, a flow channel of a mobile phase for analysis for supplying the mobile phase for analysis, a second-dimensional analytical flow channel for introducing the components trapped in the trap columns into an analytical column with the mobile phase for analysis to analyze the components, a flow channel of a mobile phase for desalination for supplying the mobile phase for desalination, and a flow channel switching mechanism for connecting the first-dimensional separation flow channel to one trap column, connecting the flow channel of the mobile phase for desalination to another one trap column, connecting the first-dimensional separation flow channel to still another one trap column, and also switching the connections of the trap columns and the flow channels, wherein the flow channel switching mechanism includes a first and second two-position valves to which one end and another end of each of the three trap columns are connected, a third two-position valve to which the first-dimensional separation flow channel, the flow channel of the mobile phase for desalination, and the flow channel of the mobile phase for analysis are connected and whose one end is connected to another end of three flow channels that are connected to the first two-position valve, and a fourth two-position valve whose one end is connected to another end of three flow channels that are connected to the second two-position valve and the second-dimensional analytical flow channel.
The flow channels are connected by valves so that the three trap columns can independently take action. By connecting the flow channels so as to perform concentration in one trap column, desalination in another one trap column, and elution in still another one trap column simultaneously, the concentration action, the desalination action, and the elution action simultaneously proceed in respective different trap columns by valves, so that it is possible to continue second-dimensional analysis with three trap columns without limiting the number of components.